Long forgotten: Eunice woodwardi Baird, 1869 (Annelida, Eunicidae) revisited, with an insight on internal anatomy

Eunice woodwardiBaird, 1869, originally described from the Ría de A Coruña (NW Iberian Peninsula), has been overlooked and never reported from the Atlantic coast of Spain after original description and the subsequent redescription of the holotype. In the present study, we revised comparatively the holotype, newly collected specimens of this species and specimens identified as Eunice vittata (Delle Chiaje, 1829) from western Mediterranean Sea. The validity of E. woodwardi is supported and previous descriptions are complemented after a throughout study of the external morphology by means of light compound microscopy and scanning electron microscopy, and that of the internal anatomy by histological sectioning and micro-computed tomography. The presence of eyes, nuchal organs, dorsal and ventral ciliary organs on parapodial cirri and paired nephridia in most segments is confirmed in E. woodwardi; the digestive tract is clearly regionalized and divided into pharynx, oesophagus, stomach, fore, mid- and hind intestine. The presence of E. woodwardi in the Ría de Ferrol is also reported, and we suggest that previous records of E. vittata in NW Iberian Peninsula should be reviewed. Eunice woodwardi is distinguished by a set of characters such as having non-articulated and non-constricted cephalic appendages, the maxillary formula, the range of branchial distribution, maximum number of branchial filaments, number of limbate and compound falciger chaetae per parapodium, the presence of an apical mucro in the guard of falciger chaetae blades and the number of teeth in pectinate chaetae. Epibiont Ciliophora on branchiae are also reported.


INTRODUCTION
Eunice Cuvier, 1817 is the most species-rich genus of the annelid family Eunicidae Berthold, 1827 and comprises 250 valid species (Read & Fauchald, 2022). It is widely distributed and can be found in soft and hard substrates from the intertidal zone to the deep sea in all oceans (Hutchings, 1986). The diagnosis of the genus was traditionally based on a combination of several morphological characters, such as the presence of three prostomial antennae, a pair of palps, a pair of peristomial cirri, and a set of chaetal types that includes: limbate, pectinate and compound chaetae, and subacicular hooks potential deterioration of the only specimen available. Anyway, it seems that E. woodwardi fell into oblivion despite Fauchald's redescription. This might also be due to E. vittata being considered as a cosmopolitan species and, therefore, it is likely that specimens of Eunice with tridentate subacicular hooks might have often been identified as E. vittata across the world. In fact, the presence of E. woodwardi in NW Spain also remained overlooked even in recent publications (e.g., Parapar et al., 1993;Parapar, Urgorri & Besteiro, 1996;Parapar & Moreira, 2009;Besteiro et al., 2018). In this context, Zanol et al. (2021) challenge the wide range of distribution traditionally attributed to several eunicids, suggesting that E. vittata might have a more restricted distribution.
In this study, we present an integrative approach to the taxonomy and anatomy of E. woodwardi in order to support the validity of the taxon. Additional anatomical information is provided after examination of the holotype and non-type material collected near the type locality; the original description and redescription were complemented with other characters not observed in the holotype, also assessing its intraspecific variability. The main features of the internal anatomy of E. woodwardi are described for the first time and the presence of epibiont ciliophorans on branchiae is also reported.

MATERIALS AND METHODS
This work is based on the study of the holotype of E. woodwardi from off A Coruña (Galicia, NW Spain), along with 34 newly collected specimens collected in the nearby Ría de Ferrol (Table 1). Selected specimens have been deposited in the Museo Nacional de Ciencias Naturales, Madrid, Spain (MNCN) and the British Museum (Natural History), London, England (NHML). For comparative purposes, specimens identified as E. vittata from several Mediterranean locations were also examined: Two specimens from Naples, Italy (type locality; Museum of Natural History of Wroclaw, MNHW), five from Venice, Italy (Zoological Museum Hamburg, ZMH), two from Banyuls-sur-Mer, France (ZMH), five from Valencia, Spain (MNCN) and four from Mallorca, Spain. Type material could not be examined because there are no longer in existence (Fauchald, 1992, p. 337). The description of E. woodwardi holotype is based on Fauchald (1992) and is complemented with our observations. Additional observations of material collected in the Ría de Ferrol and E. vittata from Naples are also provided.

Sample collection
Specimens previously identified as E. vitatta by Parapar et al. (1993) were collected in 1988 and 1989 in the Ría de Ferrol: (1) directly by hand in the rocky intertidal, (2) by pushing PVC corers into subtidal soft sediment and (3) by means of a Naturalist dredge deployed on subtidal sedimentary bottoms (see Parapar et al., 1993 for details). Additional samplings were done in subtidal soft bottoms at the Ría de Ferrol with a Van Veen grab in 2010, 2019 and 2021. Specimens were sorted from samples, fixed in 4% formalin for 24-48 h and subsequently transferred to 70% ethanol for preservation.

Anatomical study
Two specimens preserved in 70% ethanol were used for histological sectioning; they were dehydrated through a series of graded ethanol baths and clearing agent, infiltrated with paraffin and xylene in 1:1 proportion at 57 C overnight and embedded in a paraffin block. The block was sectioned with a microtome in 8 mm sections, which were placed on microscope slides, hydrated, and stained with haematoxylin-eosin, dehydrated and finally mounted on permanent slides with Canada balsam. Specimens studied with micro-computed X-ray tomography (micro-CT hereafter) were originally preserved in 70% ethanol and dehydrated in successive baths of ethanol 90% and 96%, then immersed 2 h in hexamethyldisilazane and allowed to air dry overnight (Alba-Tercedor & Sánchez-Tocino, 2011;Parapar et al., 2019) at the Estación de Bioloxía Mariña da Graña, Universidade de Santiago de Compostela, Spain (REBUSC-EBMG, USC). No staining was used. Scanning was carried out with a microtomograph Skyscan 1172 using the following parameters: 55 kV, 165 mA, unfiltered, image pixel size of 3.94 and 6.78 mm and no camera binning. Images were treated with Skyscan software: they were reconstructed with the NRecon software and cleaned with CT Analyzer software; to visualize the data, DataViewer and CTVox softwares were used. Datasets of transverse 2D images of studied specimens were uploaded at the Morphosource repository (https://www. morphosource.org/catalog/media?utf8=%E2%9C%93&locale=en&search_field=all_ fields&q=eunice+woodwardi).
Body slightly flattened dorsoventrally; most specimens whitish, original colouration not preserved in alcohol. Original colouration was observed in three specimens (Figs. 2-3, 4A, 4B). One specimen studied alive showing colouration consisting of two broad dorsal dark red bands per segment separated by bands of much lighter tone, consecutive segments separated by a dark red thin band (Figs. 2-3, 4B); bands from peristomium to posterior body end, slightly fading in tone from posterior branchial region to posterior body half depending on specimen and preservation state. One preserved specimen showing a different colouration pattern, consisting of transverse dark bands much more faded, and bands of lighter tone wider (Fig. 4A).
Prostomium nearly as wide as peristomium but shorter ( Parapodia sub-biramous. Dorsal cirri smooth, digitiform, tapering. Ventral cirri short with a digitiform tip (Figs. 7A-7E), inflated basally from about chaetiger 3 to 40; starting in chaetiger 3 in all specimens (Fig. 7C). Variation in length and width of dorsal and ventral cirri depending on specimen size, decreasing in length from anterior to posterior parapodia (Table S1). Lateral interramal red dots present in almost all body segments.
Full-size  DOI: 10.7717/peerj.13126/ fig-6 The body musculature is well developed, particularly in the pharynx and body wall (Figs. 12, 15). It is composed by circular, dorsal and ventral longitudinal and ventral oblique muscles (Fig. 15). The longitudinal musculature is divided into four bands, two arranged dorsally and two ventrally, the latter flanking the ventral nerve cord at both sides (Figs. 13A, 13C, 14C, 14D, 15).
The presence of gametes in coelomic body cavity was observed with LCM, micro-CT and HIS (Figs. 1A, 10C, 10D, 17G); specimens observed with micro-CT and HIS were collected in September-November. The specimen subjected to HIS bears gametes at an early stage of maturation and corresponds probably to a male due to the small gamete size (about 4-5 µm in diameter), high numbers and grouping shape (Fig. 17G). In anterior body half, gametes seem to be associated to the parapodial area, whereas in posterior half,  Epibiosis Ciliophorans were observed attached to the surface of branchial filaments (Fig. 19) but not showing any defined pattern of attachment or distribution.

Distribution and ecology
The type locality corresponds to Ría de A Coruña (Galicia, NW Spain). Unfortunately, Baird (1869) did not provide neither the coordinates nor depth or abiotic characteristics where this specimen was collected. Non-type specimens collected in the Ría de Ferrol were found from the intertidal to 26.5 m depth, in a wide range of bottom types, from coarse (gravel) to fine (sandy mud and mud) sediments (Table 1).
Branchiae pectinate, stem longer than dorsal cirri, present from chaetiger 3 to 27-29 (<55% of body chaetigers); first branchia with 3 filaments (Fig. 20C); up to 7 filaments per branchia in following chaetigers. Last branchiate chaetigers with one filament. Branchial filaments longer or about as long as dorsal cirri in mid-branchial region chaetigers. Parapodia sub-biramous. Dorsal cirri smooth, digitiform, tapering. Ventral cirri short with a digitiform tip, inflated basally from about chaetiger 3 to 27-29, in all specimens starting in chaetiger 3. Dorsal and ventral cirri decreasing in length from anterior to posterior parapodia (Figs. 20C, 20D). Lateral interramal red dots present in almost all body segments. Figure 16 Histological section, schematic reconstructions and FESEM micrographs of Eunice woodwardi. Nervous system and sensory organs of three specimens (Ría de Ferrol, MNCN 16.01/19147, MNCN 16.01/19148 and MNCN 16.01/19166) Two yellow neuroaciculae, one larger than the other; all neuroaciculae tapering with blunt tips, curved distally and protruding from acicular lobe, never T-shaped. Chaetae including 2-11 limbate, 1-3 pectinate, 3-23 compound falcigers and 1-5 tridentate subacicular hooks. Limbate and pectinate chaetae arranged in a bundle dorsal to neuroaciculae. Limbate chaetae elongated, marginally serrated, distally curved and tapering. All pectinate chaetae heterodont, about 0.3-0.4 times as long as limbate chaetae; number of teeth not observed, one external tooth three times as long as others. Compound falcigers ventral to neuroaciculae; shafts distally inflated and marginally serrated. Blades bidentate; proximal tooth slightly larger than distal tooth, triangular, perpendicular to blade axis; distal tooth curved dorsally; blade distal two thirds protected by elongated guard, marginally serrated; mucro absent. Number of limbate and compound falcigers Figure 17 Schematic reconstruction, FESEM micrographs of parapodia and histological section of internal anatomy of Eunice woodwardi. Four specimens (Ría de Ferrol, MNCN 16.01/19157, MNCN 16.01/19158, MNCN 16.01/19160 and MNCN 16.01/19166)  The study of the holotype of E. woodwardi and recently collected material in the NW Atlantic coast of Spain supports the validity of this species; E. woodwardi clearly differs from E. vittata mostly regarding the presence of an apical mucro in the guard of falciger chaetae blades and the number of teeth in pectinate chaetae. Furthermore, several features not previously described in E. woodwardi by Fauchald (1992) are first mentioned here. As stated before, it has not been dilucidated yet the systematic position at the genus level of all species formerly included in Eunice sensu lato. Anyway, E. woodwardi would fit into group C-1 sensu Fauchald (1970) mostly because of having tridentate subacicular hooks and first branchiae present before chaetiger 10 and ending before chaetiger 100. According to the descriptions by Fauchald (1992) and Zanol, Fauchald & Paiva (2007), the closest species to E. woodwardi are E. vittata (placed in Leodice according to Zanol et al., 2021), E. indica Kinberg, 1865 andE. unifrons (Verrill, 1900), because of sharing the same chaetal types and having also digitiform cephalic appendages separated from each other by the same distance. Eyes were not reported in all Eunice species; this character was not included in the description of E. woodwardi holotype neither by Baird (1869) nor by Fauchald (1992). However, the presence of a pair of eyes in specimens from the Ría de Ferrol is here confirmed. Therefore, eyes might have been present in the holotype and then eyes pigment would have faded after being fixed for preservation. The holotype was in ethanol for 153 years while non-type material from Ferrol was for 35 years. The maxillary formula is also described here for the first time for E. woodwardi. Several features are shared with most species of Eunice sensu lato, such as: (1) only the left MxIII is present, (2) MxI and V bear only one tooth, and (3) lack of MxVI. However, E. woodwardi differs from other species in the number of teeth of MxII, MxIII and MxIV. The most similar maxillary formula is that of E. vittata; Fauchald (1992) describes it from specimens collected near the type locality, i.e., MxI: 1+1, MxII: 9-10+9-10, MxIII: 8-9+0, MxIV: 6+8-12, MxV: 1+1. Other authors, after examining specimens of supposedly the same species from the Atlantic and Pacific (Fauvel, 1923) and Indian oceans (Day, 1967), indicated slight differences, i.e., there were more teeth in MxIV (10+13) while Şahin & Çinar (2009) reported 9+10 in MxIV for specimens from the eastern Mediterranean Sea. Thus, E. vittata would differ from E. woodwardi in having more teeth in left MxII and left MxIV, and fewer in left MxIII. Eunice indica Kinberg, 1865 also has a similar formula, i.e., MxI: 1+1, MxII: 9-11+8-11, MxIII: 8-11+0, MxIV: 7-10+13, MxV: 1+1 (Day, 1967), but this species bears more teeth in left MxII and fewer in right MxIV when compared to E. woodwardi; however, these specimens reported by Day (1967) from South Africa might correspond to a different species from E. kinbergi.
On the other hand, the presence of discocilia on branchiae had previously been reported by Heimler (1978) for Lanice conchilega (Pallas, 1766). However, Ehlers & Ehlers (1978) stated that this type of cilia represent an artificial structure; this assumption was later confirmed by Short & Tamm (1991) who stated that these artefacts were caused probably by fixation and osmotic stress and by Göbbeler & Klussmann-Kolb (2006).
Overall features of chaetal composition (limbate, pectinate and compound falciger chaetae, tridentate subacicular hooks and aciculae) in E. woodwardi are also shared with several group C-1 species. However, the presence of a mucro in compound falcigers is not shared with all species of this group such as E. unifrons and E. vittata. Fauvel (1923), Day (1967) and Campoy (1982) described compound falcigers provided with long pointed guards in specimens of what they regarded as E. vittata from the Atlantic and Pacific oceans, Indian Ocean and Mediterranean Sea, respectively. On the contrary, Fauchald (1992) examined specimens of E. vittata collected near its type locality and stated that blade guards lack mucros. Furthermore, number of chaetae per parapodium also seem to differ between E. vittata and E. woodwardi. Specimens from the eastern Mediterranean identified as E. vittata by Şahin & Çinar (2009) bear 2-5 limbate and 2-6 falciger chaetae per parapodium instead of 3-14 and 3-23 respectively as found in E. woodwardi. On the other hand, the number of pectinate chaetae per parapodium is similar in both species but they differ in number of teeth: E. vittata has up to five sensu Fauchald (1992) and E. woodwardi bears 7-9. Numbers of subacicular hooks also vary across group C-1, numbering three or more in E. woodwardi, E. indica and E. vittata and 1-2 in remaining species.
In this context, it seems that features of specimens attributed to E. vittata from across the world show much variation. Therefore, we also examined specimens from several western Mediterranean locations that were identified as E. vittata all having tridentate subacicular and blade guards lacking a distinct mucro (following Fauchald, 1992). On the one hand, the specimens of E. vittata that were collected near the type locality (Naples, Italy) also differ from E. woodwardi in having: (1) cephalic appendages with constrictions; (2) branchiae limited to fewer chaetigers (chaetigers 3-29 vs 3-40); (3) first branchia provided with 3 filaments; (4) fewer branchial filaments (up to 7 vs 9-14); and (5) tridentate subacicular hooks first present from chaetigers 21-22. On the other hand, specimens of E. vittata from other locations show differences with both E. vittata from Naples and E. woodwardi. For instance, specimens from Banyuls-sur-Mer and Mallorca bear cephalic appendages that are provided with constrictions but they differ in branchial distribution range; specimens from Valencia bear branchiae from chaetiger 3 to 36-40, while those from Banyuls-sur-Mer bear fewer branchiate chaetigers (chaetiger 3 to 26-29 and 3 to 28, respectively). They also differ in maximum number of branchial filaments: 2-3 (Banyuls-sur-Mer), 4 (Mallorca) and 7 (Valencia). The number of limbate and compound falciger chaetae per parapodium also shows variation (Tables S2, S3): 3-9 limbate and 2-13 compound falcigers (Valencia) and 3-7 and 4-13 (Mallorca), that are fewer than those found in E. woodwardi (3-14 and 3-23). Regarding subacicular hooks, E. vittata from Venice showed up to four hooks per chaetiger but never reaching up to five, while E. vittata from Valencia were characterised by having just up to two hooks but only present in the last chaetigers (107-110) of large specimens. In all, these observations support that E. woodwardi from NW Iberian Peninsula differ clearly from specimens attributed to E. vittata from western Mediterranean; the morphological variability found among the latter also suggests that there might be several species involved that share non-mucronate blade guards and tridentate hooks, not discarding the presence of exotic species as well (Zanol et al., 2021). In this context, Zanol et al. (2021) also states that E. vittata (as Leodice) and other species formerly considered as cosmopolitan might have restricted distributions once their taxonomy is clarified.

Internal anatomy
The study of several specimens with the micro-CT and through HIS has revealed for the first time the main features of the internal anatomy of E. woodwardi, such as the regionalized digestive tract or the presence of nuchal organs. The highly regionalized gut of E. woodwardi, divided into pharynx, oesophagus, stomach, fore, mid-and hind intestine, is against the opinion of Penry & Jumars (1990), i.e., that the digestive tract in carnivorous "polychaetes" is very simple, and divided only into two parts, a foregut and a hindgut. However, these authors did not include in their study neither carnivorous taxa nor any Eunice species. Anyway, we have not been able to verify whether E. woodwardi actually behave as a carnivore or not, and available information for other species is contradictory. For instance, Gaston (1987) considered E. vittata as a detritivore after examination of gut contents while Deudero et al. (2011) regarded it as a carnivore after analysing stable isotopes of carbon and nitrogen. In fact, the concept of a simple digestive tube in carnivorous species, made up of mouth, pharynx, oesophagus and intestine, had been already described by Ehlers (1868) for Eunice; however, the illustrations included in his work suggest that the digestive tract is more complex. The pharynx of E. woodwardi is ventral and may correspond to the Type 4 described by Tzetlin & Purschke (2005). We also observed a pair of small, rounded structures inside the pharynx (g, Figs. 11A, 11B), that could be similar to the glands mentioned by these authors in the same type of pharynx present in the family Dorvilleidae Chamberlin, 1919.
The arrangement of musculature, circulatory and nervous systems in E. woodwardi agrees with the typical pattern described for the annelid body plan (Beesley, Ross & Glasby, 2000;Tzetlin & Purschke, 2005). In fact, the central nervous system of E. woodwardi, composed by pairs of ganglia, almost totally fused, and located along the ventral nerve cord, is similar to that described by Ehlers (1868) for Leodice harassii (Audouin & Milne Edwards, 1833), Hofmann (1974) for Palola siciliensis (Grube, 1840) and Zanol (2010) for Eunicidae. Furthermore, different types of sensory organs such as eyes, nuchal organs and dorsal cirrus ciliary organs have been observed in E. woodwardi. Shape and location of nuchal organs are the same as described by Hofmann (1974) for P. siciliensis and Fauchald & Rouse (1997) for other members of Eunicidae. Similarly, features of dorsal cirrus organs agree with that of Leodice antennata Savigny in Lamarck, 1818 and Marphysa sanguinea (Montagu, 1813) (see Hayashi & Yamane, 1994). Ciliary areas observed below the parapodial ventral cirrus, of unknown function, might either correspond to the opening of parapodial glandular organs (Meiβner, Bick & Müller, 2012) or to another type of sensory organ like the dorsal cirrus organ.
Large glandular masses were observed in each parapodium of E. woodwardi that are probably parapodial glands associated with chaetae. These glands are similar (although smaller) to the gland-associated chaetal complex that is part of the parapodial glandular organs in Spionidae to which Meiβner, Bick & Müller (2012) suggest a secretory activity related to the chaetogenesis and tube construction.
Cuticular pores are mentioned here for the first time in E. woodwardi. Their function is likely to be excretory although the techniques used did not reveal the presence of associated glands. Similar pores were already described for Eunice by Ehlers (1868), who suggests an excretory function in L. harassii because of being connected to subcutaneous glandular masses.
Oocytes of E. woodwardi are very similar to those of P. siciliensis (see Hofmann, 1974) and, as in the latter, when they reach maturity, the longitudinal musculature in the posterior body region is much reduced and the intestinal epithelium is atrophied. In addition, this posterior region in E. woodwardi also lacks some typical elements such as blood vessels and glandular masses associated with parapodial chaetae, which is consistent with a corporal degeneration that is, in turn, directly linked with release of gametes through liberation of posterior body end. In fact, as previously mentioned, most studied specimens lack the posterior body half, and this might be due to a rough handling of the samples and/or that E. woodwardi has indeed an epitokous reproduction mode with release of the posterior body end as was reported by Wilson (1991) in other Eunice species.
The shape, size and abundance of the gametes observed through HIS in one specimen of E. woodwardi, suggests that it is probably a male with gametes in an early stage of maturation and very similar in size and shape to male gametes reported by Ouassas et al. (2015) for M. sanguinea, forming groupings (morulae sensu Ouassas et al., 2015) and accumulate in the coelomic cavity of posterior half of the body. Gonads could not be studied in our specimens, and it was not possible to assess whether the oogenesis of E. woodwardi is extraovarian or intraovarian. However, an extraovarian strategy was reported by Ouassas et al. (2015) for M. sanguinea.

Ciliophoran epibionts
Epibiosis is a widespread phenomenon and marine annelids show a variety of ecological relationships with other organisms, including different symbiotic associations (Martin & Britayev, 1998) and some have been reported as hosts (basibionts) of a large variety of other taxa (Álvarez-Campos et al., 2014). Mikac et al. (2019) states that this association can be considered as ectocommensalism because the annelid gets no harm from the epibiont. The presence of ciliophorans as epibionts on "polychaetes" has been reported previously in Onuphidae (Arias, Anadón & Paxton, 2010), Syllidae (Álvarez-Campos et al., 2014), Ampharetidae , Polynoidae and Sigalionidae (Mikac et al., 2019). Ciliophorans have been found in E. woodwardi only on branchiae but in other species can be found also in other parts, such as body surface, prostomium, mouth opening, palps, chaetae, parapodial cirri and pygidium. For instance, in the ampharetid Ampharete santillani Parapar, Kongsrud, Kongshavn, Alvestad, Aneiros & Moreira, 2018 the ciliophorans were found across the body but particularly on ciliated areas, such as the branchial surface .

CONCLUSIONS
Our examination of the holotype and additional material near the type locality confirms that E. woodwardi is a valid species and different to E. vittata. Eunice woodwardi is distinguished by the non-articulated and non-constricted cephalic appendages, the maxillary formula, the range of branchial distribution, the maximum number of branchial filaments, the presence of an apical mucro in the guard of falciger chaetae blades, and the number of teeth in pectinate chaetae. Eunice woodwardi is at least present in the rias of A Coruña and Ferrol, and we suggest that previous reports of E. vittata from the NW Iberian Peninsula should be reviewed. The integrative use of different anatomical techniques for the study of the internal anatomy has confirmed previous observations in the genus Eunice sensu lato and allowed reporting some previously unknown structures such as the ciliated areas located below ventral cirrus. The presence of ciliophoran epibionts on branchiae is also reported.